1) Studies with Ca. We have measured total Ca content in intact red cells using both Ca45 and total Ca in dry ashed red cells and find that in each case equilibrium is achieved within two minutes and the results are identical with both isotope and total Ca. Measurement of binding constants of Ca to the external membrane surface by Scatchard analysis has been carried out under physiological conditions and our results have been compared with binding constants obtained under quite different conditions by other workers. We have begun experiments to measure Ca binding to the inside of the membrane using inside-out vesicles prepared by the technique of Steck et al. Preliminary measurements have been made of binding constants of the Ca-membrane complex to allow comparison with the outside of the membrane. Treatment of the membrane with phospholipases and various protein reagents may allow us to identify the binding sites. We plan to study the kinetics of the Ca-membrane reaction by use of a recently modified temperature jump apparatus which will enable us to study chemical reactions with a resolution of better than 5 microsec. 2) Studies with phospholipid vesicles. For the work with vesicles, phospholipd vesicles have been prepared by the method of Huang. These vesicles are homogeneous in size and well defined in their properties. As expected, ANS (1-Anilino-8-Naphthalene Sulfonate) binds to these vesicles but can be displaced by competition from other solutes. We have studied ANS displacement by the homologous series of amides from formamide through valeramide and found, as expected, that the displacement increases with increasing chain length. We have also studied the temperature dependence of the amide-vesicle interaction and will compare these results with findings of similar interactions with human red cell membranes.